gel filtration (gf)wolfson.huji.ac.il/purification/course92632_2014... · 2019. 11. 25. · 0 10 20...
TRANSCRIPT
1
Gel Filtration (GF)
Size Exclusion
Chromatography (SEC)
2
Gel Filtration chromatography (GF)
Principles of GF
Fractionation range
Parameters for resolution optimization
Use of GF: MW/oligomeric state – purification – buffer
exchange - QC
Examples - Troubleshooting
SEC-MALS
3
What is gel filtration?
• SEC is the most powerful chromatography technique for
obtaining reliable information about the size of
biomolecules under native conditions
• Separates molecules according to their hydrodynamic ratio
(size, conformation & oligomeric state)
• Different fractionation ranges: beads with pores of well-
defined sizes
• Mobile phase: almost all kind of buffers
4
Gel structure
AgaroseDextran
A hypothetical structure for Superdex
Different columns with beads of defined porosity: fractionation range
The degree of cross-linking determines the size of the pores and therefore the
fractionation range of the resin
SEC is not an adsorption technique (unlike all other chromatographic
procedures).
Void volume Vo
Volume of the gel matrix Vs
Pore volume Vi
How does it work?https://www.youtube.com/watch?v=oV5VB5kO3tQhttps://www.youtube.com/watch?v=E3z1wIImvHIhttps://www.youtube.com/watch?v=rPRbqYWlSEo
Smaller molecules spend longer in the
pores and elutes later
Larger molecules spend less in the pores and elutes
sooner
6
Terms and explanationsVo= Void volume: volume of the solution outside the beads, or elution from very large molecules
Ve = the volume from the time the protein is placed until it appears in the effluent
Vi = volume of the solution inside the beads = Vc - Vs - Vo
Vc = Total (geometric) volume of the column
Vt = Elution volume for very small molecules
2 3
Void volume Vo
Volume of the
gel matrix Vs
Pore volume Vi
1
Vo
Ve
Vt
Vc
7
Steric exclusion
Molecules are excluded from the gel bead to different extents according to their sizes.
Gel bead
Largest molecules - excluded from pores, travel with the mobile phase, elute rapidly from column• The volume at which large molecules elute is called the void volume, Vo (same as the volume of solution that surrounds the beads)
Smallest molecules – enter the pores of the beads, are included in the matrix and retarded in their movement, spend most of the time in the stationary phase, elute last• The volume at which small molecules elute corresponds to Vt (total volume of solution surrounding (Vo) and inside the beads, Vs) Vt = Vo + Vs
Intermediate size molecules – spend different amounts of time both inside and outside the beads (partition between the mobile and stationary phase)• The volume at which intermed.molecules elute is called the elution volume (Ve) and depends on the partition of the molecule between the Vo and Vs which is proportional to the distribution coefficient (K) Ve = Vo + KVs
8
Column calibration: MW extrapolation of unknown molecule
Kav 1
0log (Mr)
• Run MW standards and determine the elution volume for each (globular proteins)
• Calculate Kav values
• Plot log (Mr) for each standard against the calculated Kav
• Selectivity curve is usually moderately straight over the range Kav=0.1 to Kav=0.7
ot
o
VV
VVeavK
Extrapolate MW of globular proteins according to Ve
Limitations: protein is not globular; interaction with the resin
Denatured proteins
Kav 1
0.7
0.1
log (Mr)
Native proteins
Shape effects
Elution volume depends:
•Native, globular proteins
•Partially folded molecules
•Oligomeric state (monomer, dimer, trimer…soluble aggregate)
•Proteins inside detergent micelle: MW of protein + MW of micelle
10
How to choose GF type
Selectivity – fractionation range Kav 1
log (Mr)
Molecules with different shapes have different
selectivity curves
Linear polysaccharides
Globular proteins
RESIN FR Glob Prot FR Dextrans
Sephacryl S100 1-100 kDa ND
Sephacryl S200 5-250 kDa 1-80 kDa
Sephacryl S300 10-1500 kDa 2-400 kDa
Sephacryl S400 20-8000 kDa 10-2000 kDa
Protein 1: 30kDa
Protein 2: 80kDa
Vo Vt
11
Gel Filtration chromatography (GF)
Principles of GF
Fractionation range
Parameters for resolution optimization
Use of GF: Purification – buffer exchange – QC -
MW/oligomeric state – Protein/protein interaction
Examples - Troubleshooting
SEC-MALS
12
13
Efficiency
Efficiency depends on
Particle size of matrix (particle size distribution)
Packing quality of the column
Sample: volume, purity, concentration and viscosity
Flow rate (important only for big particle size)
Tubing diameter, tubing length and flow path volume
Use minimal tubing length
Shorter distance from injection valve to column
14
Peak width depends on particle size
Superdex Peptide 13-15 µm Superdex 30 prep grade 24-44
µm
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Retention time (min)
15
1 x Superdex® Peptide HR 10/3000 2 x Superdex® Peptide HR 10/30
Resolution depends on column length
Increasing column length increases resolution
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40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 ml
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Superdex® Peptide 60 x 1.6cm ~ 120ml Superdex® Peptide 100 x 1.6cm ~ 200ml
SUMO-Atox1
SUMO-Atox1
SUMO
SUMO
Atox1
Atox1
Michal Shoshan from Edith Tshuva lab.
16
Column: Superdex® Peptide HR 10/30
Resolution depends on sample volume
25µl
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Retention volume (ml)200 µl
400 µl
00.010.020.030.040.050.060.07
0 10 20 30
Retention volume (ml)
00.020.040.060.080.1
0.12
0 10 20 30
Retention volume (ml)
17
Increasing resolution
Choose appropriate fractionation range
Increase column volume (Connect columns in tandem)
Reduce the flow rate
Change to a gel with smaller beads (higher efficiency)
Reduce the sample volume / protein quantity
Volume ~0.5-4% times the expected sample volume
Sample volume can be increase if resolution is OK
Check the column efficiency
Clean and/or re-pack
18
SEC Applications
Group separations: Desalting, Buffer exchange, Removing
reagents (replace dialysis)
Purification of proteins and peptides: complex samples,
monomer/dimer
QC: Size estimation. Size homogeneity: oligomeric state.
Impurities. Stability
Protein-Protein Interaction
19
HSA NaCl
volume
Desalting proteins
Desalting in a simple column
Column:
Sample:
Buffer:
PD-10
HSA, 25 mg
NaCl 0.5M
Volume for desalting: up to 25% column volume
20
Desalting / Buffer Exchange / Group separation
Adjusting pH, buffer type, salt
concentration during sample
preparation, e.g. before an assay.
Removing interfering small molecules:
EDTA, Gu.HCl, etc
Removing small reagent molecules,
e.g. fluorescent labels, radioactive
markers
Alternative to dialysis or to diafiltration
(ultrafiltration at constant retentate)
Gravity Desalting Columns
Multi Spin
Desalting Columns
FPLC Desalting Columns
Spin Desalting Columns
21
HiTrapDesalt10ml001:1_UV1_280nm HiTrapDesalt10ml001:1_Cond HiTrapDesalt10ml001:1_Fractions HiTrapDesalt10ml001:1_Inject HiTrapDesalt10ml001:1_Logbook
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HiTrapDesalt10ml002:1_UV1_280nm HiTrapDesalt10ml002:1_Cond HiTrapDesalt10ml002:1_Fractions HiTrapDesalt10ml002:1_Inject HiTrapDesalt10ml002:1_Logbook
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Waste 17 18 Waste
Desalting in the presence of buffer + 250mM NaCl
Desalting in the presence of buffer + 100mM NaCl
OD 280nm
Conductivity
Use buffer that avoid protein precipitation
Gali Prag
22
Fractionation of multiple components
Separate multiple components in a sample on the basis of differences on
their size
Best results with samples that contains few components or partially purified
samples (polishing step) : Not recommended for proteins with close MW
Limited sample volume (0.5-4% of total column volume). Not so suitable if
the sample volume is large. Volume can be increase if resolution is still OK
(scale up)
Flow-rate limitation : Time consuming
Removes higher oligomeric states and other aggregates
Protein elutes with equilibration buffer (important for storage or buffer exchange)
170726TIMPSuperdex75Prep120ml001:10_UV1_280nm 170726TIMPSuperdex75Prep120ml001:10_UV2_260nm 170726TIMPSuperdex75Prep120ml001:10_UV3_220nm 170726TIMPSuperdex75Prep120ml001:10_Cond 170726TIMPSuperdex75Prep120ml001:10_Fractions 170726TIMPSuperdex75Prep120ml001:10_Inject
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F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Secreted Yeast TIMP9 after IMACSuperdex 75 60x1.6cm ~120ml
Jason ShiranYulia Shifman lab
Separating dimer and oligomers from monomer
24
Column size- Sample preparation
Desalting and other group separations
Column volume: four times the expected
sample volume
Column length is not so important
Preparative separation
Column volume: 0.5-4% times the expected
sample volume
Sample volume can be increase if resolution
is OK
Column length: 30-100 cm or more
(depends of the resolution: higher length
higher resolution)
How to reduce volume or concentrate your
sample
• Ultrafiltration
• Lyophilization
• Ammonium Sulfate Precipitation (or similar)
• Reverse elution (bind to absorption column
like IEX, HIC, IMAC; and reverse elution)
• Dialysis vs hygroscopic environment (glycerol, PEG, Sephadex etc)
• Others
25
Quality control: QC size estimation / oligomeric state / impurities / stability
Monitor protein prep quality
In analytical SEC, the sample volume should be approximately 0.3% of the bed
volume to achieve optimal results.
Complementary information to PAGE-SDS
Gives an estimate of molecular size in native solution
Un-native solution: Guanidine HCl, urea, detergents, etc. Precision is not so good as PAGE-SDS
For exact MW use SEC-MALS
Oligomeric state of the protein / homogeneity / complex. Aggregation profile.
Detect presence of impurities.
Identify protein interaction partners and interaction conditions
120402HLTVirE2Superose12prepEndwash001:1_UV1_280nm 120402HLTVirE2Superose12prepEndwash001:1_UV2_260nm 120402HLTVirE2Superose12prepEndwash001:1_UV3_220nm 120402HLTVirE2Superose12prepEndwash001:1_Fractions 120402HLTVirE2Superose12prepEndwash001:1_Inject 120402HLTVirE2Superose12prepEndwash001:1_Logbook
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
HLT-VirE2Purification from 200ml cell
culture. IMAC purification and Preparative Gel Filtration
Superose 12 60x 1.6cm = 200ml
Michal Maess from Assaf Friedler lab.
SEC gives different and complementary information to PAGE-SDS
Size separation in native or un-native conditions
QC: monitoring size-homogeneity changes during storage or stress conditions
AggregationEvaluate tendency to aggregate and
quantity aggregates
DegradationEvaluate tendency to degradetion and
quantity of degraded forms
The trend is towards smaller particles of < 2 μm, with the use of
ultra high-performance liquid chromatography (UHPLC) systems for
even faster separations in high-throughput mode.
But:
• Very high back pressure
• Demands specific equipment
• Loss of resolution due to dead volumes
• Heat generation and shear stress at high flow rates could affect
proteins
A new trend: UHPLC (Ultra high-performance liquid chromatography)
29
Gel Filtration chromatography (GF)
Principles of GF
Fractionation range
Parameters for resolution optimization
Use of GF
Troubleshooting
Examples
SEC-MALS
30
Troubleshooting
Lower yield than expected
Protease degradation of the protein
Adsorption to filter, valves or top of the column
Non-specific adsorption
Sample precipitate
MW of protein is not as expected
Oligomerization state of the protein is different
Protein bounds to another protein or complex
Unfolded or naturally unfolded protein
Protein has changed during storage
Ionic or Hydrophobic interactions with the matrix
Protein precipitate
Very broad peak elution
Different oligomeric states or protein aggregation
Sticky protein
Non specific adsorption to matrix
Protein is part of complex with different sizes
Overloading
Peak of interest is poorly resolved
Sample volume is too high
Column length is not enough
Poor selectivity or efficiency of the column
Flow rate too high
Column is dirty or not well packed
Viscous sample
11/25/201931
Case study: HLT-p53CT
Capture: IMAC Affinitystart with pellet of 1.5L culture Ni-Sepharose FF 14ml
HLTp53CTNiNTA16ml004:1_UV1_280nm HLTp53CTNiNTA16ml004:1_UV2_260nm HLTp53CTNiNTA16ml004:1_Conc HLTp53CTNiNTA16ml004:1_Fractions HLTp53CTNiNTA16ml004:1_Inject HLTp53CTNiNTA16ml004:1_Logbook
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F4 Waste 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Load + 10cv 0%B + 3cv 8%B + 4cv 15%B + 4cv 100%B
POOL 17-22: 3.5OD x 35ml ~ 276mg
Ronen Gabizon from Assaf Friedler lab.
11/25/201932
Case study: HLT-p53CT
IntermediateCation Exchange
After TEV protease cleavage ON 4ºC
SP-Sepharose FF 5ml
HLTp53CTHiTrapSP5mlml005:1_UV1_280nm HLTp53CTHiTrapSP5mlml005:1_UV2_260nm HLTp53CTHiTrapSP5mlml005:1_Cond HLTp53CTHiTrapSP5mlml005:1_Conc HLTp53CTHiTrapSP5mlml005:1_Fractions HLTp53CTHiTrapSP5mlml005:1_Inject HLTp53CTHiTrapSP5mlml005:1_Logbook
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F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
33
Case study: HLT-p53CT
Polishing SEC columnColumn: Sephacryl S100 prep. 960 x 26mm (~500ml) 6ml/fract.
Ni column – TEV protease cleavage ON – dilution – CEIX – concentration & GF
HLTp53CTSephacrylS100of500ml004:1_UV1_280nm HLTp53CTSephacrylS100of500ml004:1_UV2_260nm HLTp53CTSephacrylS100of500ml004:1_Fractions HLTp53CTSephacrylS100of500ml004:1_Inject HLTp53CTSephacrylS100of500ml004:1_Logbook
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150 200 250 300 350 400 ml
F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
Fractions around 23 and 32 are higher MW impurities
POOL 7-14
Ronen Gabizon from Assaf Friedler lab.
Increasing resolution Example: Pegylated protein
120617M1605Superose12Anal001:1_UV1_280nm 120617M1605Superose12Anal001:1_UV2_260nm 120617M1605Superose12Anal001:1_Fractions 120617M1605Superose12Anal001:1_Inject 120617M1605Superose12Anal001:1_Logbook
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120618Superose12Prep3columns500ml001:1_UV1_280nm 120618Superose12Prep3columns500ml001:1_UV2_260nm 120618Superose12Prep3columns500ml001:1_Fractions 120618Superose12Prep3columns500ml001:1_Inject 120618Superose12Prep3columns500ml001:1_Logbook
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2 3 4 5 6 7 8 9 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93Superose 12 analytical 30 x 1cm = 23ml column
Load : ~1mg proteinSuperose 12 preparative
3 tandem columns 250 x 1.6cm = 502ml columnLoad : ~25mg protein / 5ml
Vo
123
How can we get better separation between 2 and 3 ??
Can we scale-up protein loading to separate 1 from 2 and 3 ??
35
250
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50
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25
20
15
10
Before 6 7 M 8 9 11 13 15 17 19 21
23 25 27
Without DTT / boilingPool fractions 8-17
Results Fitting
Protein Molar Mass 1 Protein Molar Mass 2 LS UV RI
volume (mL)
9.0 10.0 11.0 12.0 13.0 14.0
Mo
lar
Mas
s (g
/mo
l)
41.0x10
51.0x10
61.0x10
160216 Darpin:10_UV1_280nm 160216 Darpin:10_UV2_260nm 160216 Darpin:10_Fractions 160216 Darpin:10_Inject 160216 Darpin:10_Logbook
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60.0 80.0 100.0 120.0 ml
cync
y to
wn
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Mer
lin 1
Cin
cy T
own
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Waste 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Waste 32 33 34 35 36 37 38 39 40 41 42 43 Waste 45 46 47 48 49 50 51 52 53 54 55 Waste
Peak1:Total mass = 850±30 kDaProtein mass = 440±20 kDaRation = ~1:1Peak2:Total mass = 85±5 kDaProtein mass = 2.6±0.2 kDa
Protein MW (kDa) Ve (ml)
Thyroglob 669 9.76
Ferritin 440 10.95
Catalase 232 12.66
BSA 67 14.22
Chymotr 25 17.52
Peak 1 9.6
Peak 2 12.6
CMV-Viral Glycoprotein Extracellular Expression in Insect Cells
100kDa Ultrafiltration and SEC purification in tandem Superose 12 & Superdex 200 100 x 1.6cm each ~ 400ml
total SEC-MALS: Superdex 200
analytical column
36
Complex formationExample: Leptin and Leptin Receptor
Superdex75prep002:1_UV3_220nm Superdex75prep002:1_Fractions Superdex75prep002:1_Inject Superdex75prep002:1_UV3_220nm1Superdex75prep002:1_UV3_220nm2 Superdex75prep002:1_Logbook
20.0
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100 150 200 250 ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3031 32 33 34 35 Waste
Superdex 75 160x1.6cm column - Buffer: 20mMTrisHCl pH8.0 50mMNaCl 0.02%NaN3
Receptor alone
Leptin alone
Complex: Leptin + Receptor
37
Why choose gel filtration?Advantage
Separates by size
Complementary to IEX and HIC
Very gentle, high yields
Works in any buffer solution
Removes aggregates
Fast for buffer exchange
Mostly use in a final polishing step
Mandatory for QC
Complementary results than PAGE-SDS
Disadvantage
Limited sample volume
Poor resolution in a complex mixture
Flow-rate limitation – time consuming
Sample is diluted during elution
Poor selectivity compared with SDS-PAGE
Not efficient in capture or intermediate
steps
38
HTL435aaSuperdex200prep500mlB005:11_UV3_220nm HTL435aaSuperdex200prep500mlB005:11_Logbook
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Case study: Native Unfolding Protein
HTL - A natively unfolded proline-rich domain in ASPP2 that regulates its protein interactions by intramolecular binding to the Ank-SH3 domains.
Shahar Rotem et al. JBC Friedler lab
660 440 232 156 67 kDa
HTL 435aa
FoldIndex©: a simple tool to predict whether a given protein
sequence is intrinsically unfolded. Jaime Prilusky, Clifford E.
Felder, Tzviya Zeev-Ben-Mordehai, Edwin Rydberg, Orna Man,
Jacques S. Beckmann, Israel Silman, and Joel L. Sussman, 2005,
Bioinformatics.
435 aa
MW: 49.7kD
Superdex 200 prep. 100x2.6cm : trimer ??
Analytical ultracentrifugation: monomer
Size exclusion chromatograph in line with multi angle light scattering ,
added value to characterize proteins mass and shape in native solution
conditions
Calculating Mw and radius from the light scattering equations – much
more accurate.
Calculate the Mw during the elution peaks- detect homogeneity
sample.
Detect low amount of aggregation – large molecules amplify the
intensity of LS.
Useful for protein/protein or protein/ligand interaction
Size Exclusion Chromatography - Multi Angle Light Scattering
SEC-MALS
• The intensity of the radiated light depends on the magnitude of the dipole
and the macromolecule concentration.
• When a laser light hits a macromolecule, the electric field of the light
induces an oscillating dipole that re-radiates light.
Multi Angle Light Scattering (MALS)
or
Small molecule/ low concentration
Big molecule/high concentration
LS in
ten
sity
I - intensity of scattered light
c – concentration
M – mass
dn/dc = refractive index increment
2
scattered
dc
dnMcI
I
Multi Angle Light Scattering (MALS)
Quasi Elastic Light Scattering (QELS)
Dynamic Light Scattering (DLS)
• Measures by random motion of the macromolecules.
• The fluctuations are related to the rate of diffusion (D) which is related to
the radius of the molecule (R).
• Stokes-Einstein equation:
R- radiusk- Boltzmann constantT-temperatureD- diffusion coefficientη- viscosity
Mini DAWN TREOS Wyatt technology
Triple-angle MALS, 60mW Laser Mass up to 100,000 kDa
In line with refractive index detector: measuring concentration in a
universal way
The LS detector uses the UV signal from the FPLC to
measure protein concentrations
Mini DAWN TREOS Wyatt technology
SEC-ultraviolet (UV) / LS /refractive index (RI)
approach• During elution from the column the molecules are introduced to the MALS system where
light scattering at several angles is measured, together with the dynamic light scattering and
the refractive index signals.
• The LS detector uses the UV signal from the FPLC to measure protein concentrations
• Refractive Index detector (RI ) measures the RI change of an molecule relative to the solvent
• RI gives us the possibility to calculate total concentration of all type of molecules in an
universal way, comparing it to a known reference without the need of UV
• So, we measure in this way concentration according to UV and according to RI
• This allows calculations of the molar masses and hydrodynamic radii for each peak eluted
during chromatography
• Moreover, we can extrapolate protein mass from the total mass of
the molecule
Static Light Scattering:
RMS Radius or Rg• mass averaged distance of each point in
a molecule from the molecule’s center of gravity.
• lower limit 10 nm
Dynamic Light Scattering:
Rh or Hydrodynamic Radius • radius of a sphere with the same diffusion
coefficient as “our” sample.
• lower limit ~ 0.5 nm
Rh
Radius of gyration vs. hydrodynamic radius
8 9 10 11 12 13
0.0
0.5
1.0
No
rmal
ized
ab
sorb
ance
at
28
0 n
m
Volume (ml)
10.45 ml (~45 kDa)
An intrinsically disordered protein (17 kDa)
SEC alone
Calculating mass of intrinsically disordered proteins
Predicted elution volume according to calibration curve
8 9 10 11 12 13
0.0
0.5
1.0
LS
UV
Volume (ml)
No
rmal
ized
inte
nsi
ty
17.1 0.5kDa
An intrinsically disordered protein (17 kDa)
102
103
104
105
106
Mo
lar
mas
s (D
a)
Calculating mass of intrinsically disordered proteins
Predicted elution volume according to calibration curve
Ve: 10.45 – 45kDa according to calibration curve
LS is very sensitive for aggregates
5 6 7 8 9 10 11 12
0.0
0.2
0.4
0.6
0.8
1.0 LS
UV
Volume (ml)
No
rma
lize
d in
ten
sity
102
103
104
105
106
107
108
109
1010
31.8 0.6 kDa Mo
lar
ma
ss (
Da
)
Monomer (99.7%)Aggregate (0.3%)
Protein aggregation induced by a specific molecule
8 10 12 14 16
0.0
0.5
1.0
Alone
+ M (low conc.)
+ M (high conc.)
Elution volume (ml)
No
rmal
ized
UV
inte
nsi
ty
105
106
107
108
Mass (D
a)
7 8 9 10 11
0.0
0.5
1.0 WT
L344A
Elution volume (ml)
No
rmal
ized
UV
inte
nsi
ty
P53 CTD (11 kDa) WT & L344A mutant
6050
40
30
20
Mass (kD
a)
10
P53 CTD oligomerization
WT- tetramer, L344A mutation inhibits P53 CTD oligomerization
Studying protein modifications
• Use to study protein modification such as glycosylation and pegylation.
• Can be used to characterize number of modifications.
• Can be used to study structural changes in modified proteins.
Mo
lar
mas
s
Volume Volume
Hyd
rod
ynam
ic r
adiu
s
50
51
Molar masses for two distinct ADC (antibody-drug conjugates)
formulations are determined using SEC-MALS analysisWYATT Technology
5 10 15 20 25 30
0.0
0.5
1.0
Polymer sample
LS
RI
Mass
Elution volume (ml)
No
rmal
ized
in
ten
sity
102
103
104
105
106
107
108
Mass (D
a)
Studding polymers using MALS
Downstream application in industry –measure aggregation percent
53
Preparative column:
Fast SEC-MALS experiment
5 6 7 8 9 10 11 12
0.0
0.2
0.4
0.6
0.8
1.0
Volume (ml)
No
rma
lize
d in
ten
sity
Analytical SEC-MALS :
0.01%0.5%
5%
UV
Ab
sorb
ance
Elution time (min)
Static light scattering to characterize membrane proteins in detergent solution
D.J. Slotboom et al. / Methods 46 (2008) 73–82
For the determination of the
absolute molecular mass of
membrane proteins in
protein/detergent/lipid micelles
The size exclusion column is used to physically separate
aggregates/empty micelles from the protein of interest, and
to ensure that the protein is dissolved in the correct buffer.
The elution volume is not included in the calculations.
The technique provides very similar information as
sedimentation equilibrium centrifugation, with similar
accuracy of the determined molecular masses.
Summary of SEC-MALS• SEC-MALS is a useful tool to determine protein shape and mass,
characterize oligomerization/aggregation and verify protein purity.
Very sensitive to presence of aggregates
• Additional information: modifications (glycosilation, pegylation, etc)
• Choose a good column for best separation of the sample for
achieving accurate results.
• Limitation: can detect low amount of large macromolecules but
needs high concentration of small macromolecules
• Requires longer equilibration time
56
Some molecules are highly hydrophobic, making them incompatible with fractionation via
size-exclusion chromatography (SEC).
Field flow fractionation (FFF) separates macromolecules and nanoparticles by size without
a stationary phase, eliminating most of the non-ideal surface interactions prevalent in SEC.
In an Asymmetric-Flow FFF separation channel,
macromolecules and nanoparticles are gently
pushed against a semipermeable membrane by
crossflow.
Smaller particles diffuse back up towards the
center of the channel.
Laminar channel flow induces a parabolic flow
velocity profile, causing smaller particles to elute
earlier.
Field flow fractionation (FFF)
Literature GE-Healthcare: Gel Filtration - Principles and Methodshttp://wolfson.huji.ac.il/purification/PDF/Gel_Filtration/GE_Size_Exclusion_Chromatography_Handbook.pdf
GE-HEALTHCARE Packing of Gel Filtration column and column evaluation (movies)http://wolfson.huji.ac.il/purification/Purification_Protocols.html#GF
TOSOH: GelFiltrationhttp://wolfson.huji.ac.il/purification/PDF/Gel_Filtration/TOSOH_GF.pdf
• Burgess, R.R. A brief practical review of size exclusion chromatography: Rules of thumb, limitations, and troubleshooting. Protein Expr Purif. 150, 81–85, doi: 10.1016/j.pep.2018.05.007 (2018)
• Chakrabarti, A. Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography. Antibody Engineering. doi: 10.5772/intechopen.73321 (2018).
• Mogridge, J. Using light scattering to determine the stoichiometry of protein complexes. Methods Mol Biol. 1278, 233–238, doi: 10.1007/978-1-4939-2425-7_14 (2015).
• Slotboom, D.J., Duurkens, R.H., Olieman, K., Erkens, G.B. Static light scattering to characterize membrane proteins in detergent solution. Methods. 46 (2), 73–82, doi: 10.1016/j.ymeth.2008.06.012 (2008).
• Miercke, L.J., Robbins, R.A., Stroud, R.M. Tetra detector analysis of membrane proteins. Curr Protoc Protein Sci. 77, 29.10.1-30, doi: 10.1002/0471140864.ps2910s77 (2014).
• Minton, A.P. Recent applications of light scattering measurement in the biological and biopharmaceutical sciences. Anal Biochem. 501, 4–22, doi: 10.1016/j.ab.2016.02.007 (2016).
• WYATT Technology Literature https://www.wyatt.com/library.html Bibliography https://www.wyatt.com/library/bibliography.html
Literature SEC-MALS• Some, D., Amartely, H., Tsadok, A., Lebendiker, M. Characterization of Proteins by Size-Exclusion Chromatography
Coupled to Multi-Angle Light Scattering (SEC-MALS). J. Vis. Exp.(148), e59615, doi:10.3791/59615 (2019).
• Folta-Stogniew, E., Williams, K.R. Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J Biomol Tech. 10 (2), 51–63, at <https://www.ncbi.nlm.nih.gov/pubmed/19499008> (1999)
• Kendrick, B.S., Kerwin, B.A., Chang, B.S., Philo, J.S. Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states. Anal Biochem. 299 (2), 136–146, doi: 10.1006/abio.2001.5411 (2001)
• Folta-Stogniew, E. Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. Methods Mol Biol. 328, 97–112, doi: 10.1385/1-59745-026-X:97 (2006).